Means for the inhibition of anti-beta1-adrenergic receptor antibodies

ABSTRACT

Embodiments of the present invention provide for novel peptides of use for detection and/or inhibition of anti-β1-adrenergic receptor antibodies. Certain embodiments concern uses of cyclic and/or linear peptides. In other embodiments, the present invention relates to novel peptides of use in diagnostic and/or pharmaceutical compositions. Some embodiments concern diagnosing and/or treating cardiac conditions. Cardiac conditions of the instant invention can concern infectious heart disease, non-infectious heart disease, ischemic heart disease, non-ischemic heart disease, inflammatory heart disease, myocarditis, cardiac dilatation, idiopathic cardiomyopathy, idiopathic dilated cardiomyopathy, immune-cardiomyopathy, heart failure, and any cardiac arrhythmia condition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority to U.S. Pat.No. 8,187,605 previously application Ser. No. 11/910,258, filed Oct. 1,2008, which claims priority to PCT Application No. PCT/EP2006/002977,filed Mar. 31, 2006. Both applications are incorporated herein byreference in their entirety for all purposes.

The present invention is related to peptides, their use in the detectionand inhibition of anti-β1-adrenergic receptor antibodies and diagnosticagents and pharmaceutical compositions containing the same.

Dilated cardiomyopathy (DCM) is a severe cardiac disease of young adultswhich will result in continuous decline of cardiac function if nottreated. Said decline is based on a reduced cardiac function going alongwith dilatation of the heart muscle. In about 30% of the cases dilatedcardiomyopathy is of genetic origin, about 10% of the cases are causedby toxic substances such as alcohol and chemotherapeutics and theremaining 60% of the cases are caused by acute or chronic infection ofthe heart muscle and concomitantly occurring immunological secondaryreactions. Ultimately, heart transplantation is the means of choice forthe treatment of dilated cardiomyopathy. Without treatment the patientis facing an untimely death (Richardson et al. (1996) Circulation, 93,841-842).

In idiopathic dilated cardiomyopathy which is characterised by a loss ofheart function without defined aetiology, a correlation of heart diseaseand autoimmune reactions against various myocardial antigens was found,such as autoantibodies against the heavy chain of myosin, laminin andthe ADP/ATP transporter (Schulze et al. (1990) Circulation, 81,859-869). However, there was no evidence that any of these antigens isrelevant in terms of pathogenesis. In contrast thereto, functionallyactive antibodies against cardiac β1-receptors have been shown to playan important role in the pathogenesis of dilated cardiomyopathy.Immunological analysis have shown that the second out of a total ofthree extracellular domains of the β1-adrenergic receptor exhibits botha T-cell epitope and a B-cell epitope (Hoebecke et al. (1994), MethodsNeuroscie. 25, 345-365) and meets thus the criteria for an auto-antigenin accordance with the first Witebsky's postulate.

In order to provide experimental evidence that this auto-antigen isrelevant for the etiology of dilated cardiomyopathy, it was tried toinduce immune cardiomyopathy in accordance with the second Witebsky'spostulate. In a rat model experimental evidence was provided thatstimulatory anti-β1-AR antibodies against the second extracellulardomain of the β1-adrenergic receptor are involved in the etiology ofdilated cardiomyopathy (β1-EC_(II)) (Jahns et al. (2004) J. Clin.Invest. 113, 1419-1429). The β1-EC_(II)-/GST fusion protein used showed100% homology between human and rat. Each of the animals immunizedagainst β1-EC_(II) developed stimulatory anti-β1-antibodies and severeprogressive dilatation and pump function failure of the left ventriclecorresponding to immune cardiomyopathy which could be detected byechocardiography and was confirmed by invasive measurements andhistologic analysis of the animals. In order to simulateβ1-autoantibodies the serum of β1-antibody positive animals wastransferred intravenously to genetically identical rats every fourweeks. Again, a slowly progressive dilated immune cardiomyopathy wasobserved nine months after serum transfer as evidenced byechocardiography and as also confirmed by invasive measurements andmorphological and histological studies of the hearts of the transferanimals. Using this approach, for the very first time it was shown thatβ1-antibody-induced immune cardiomyopathy can be transferred and meetsthus the classical third Witebsky's postulate for an autoimmunepathogenesis of dilated cardiomyopathy (Freedman & Lefkowitz (2004) J.Clin. Invest. 113, 1378-1382).

In addition to the animal studies blood samples from patients wereanalyzed for β1-receptor antibodies. Jahns et al. detectedauto-antibodies against recombinant human β1-receptors in about 30% ofall patients suffering from cardiac failure and idiopathic dilatedcardiomyopathy. These patients exhibit a significantly decreased cardiacfunction compared to patients without such β1-receptor antibodies (Jahnset al. (1999), Circulation 99, 649-654). The stimulatory effect of theantibodies could be abolished by the cardioselective β1-receptor blockerbisoprolol. In view of this, Jahns et al. (Jahns et al. (1999),Circulation 99, 649-654; Jahns et al. (2000) JACC. 36, 1280-1287; Jahnset al. (2004) JCI. 113, 1419-1429) suggested to use β1-receptorantagonists such as bisoprolol as inhibitors of the interaction betweenanti-β1-adrenergic receptor antibodies and the native recombinant andnon-recombinant β1-adrenergic receptors.

An alternative therapeutic approach for the treatment of dilatedcardiomyopathy is the removal of the antibodies from the circulation ofpatients with functionally active and potentially harmful β1-receptorantibodies using immunoadsorption or immunoapharesis. In such aprocedure, the blood of the patient is passed through columns removingthe auto-antibody against β1-adrenergic receptors using a matrix couplednon-receptor homologous peptide or by inspecifically removing antibodiesof the IgG subclass by binding the antibodies to protein A columns or tomatrix coupled goat-anti-human IgG antibody. Such procedure, however, isboth time and labour consuming. Additionally, due to the unspecificelimination of all immunoglobulins from the blood of the thus treatedpatients, numerous draw-backs arise such as an imbalanced immune systemand immune response. Nevertheless it seems that antibody-positive DCMpatients have at least a short term benefit from such treatment.

The problem underlying the present invention is thus to provide furthermeans for the inhibition of anti-β1-adrenergic receptor antibodies. Moreparticularly, the problem underlying the present invention is to providemeans for the inhibition of anti-β1-adrenergic receptor antibodies.

The problem underlying the present invention is solved in a first aspectby a peptide selected from the group comprising

a) a cyclic peptide of formula I:

(SEQ ID NO: 3) cyclo(Ala-x-x-x-x-x-x-x-x-x-Cys-x-x-x-Pro-x-Cys-Cys-x_(k)-Gln), (I)whereby k is any integer from 0 to 6, preferably any integer from 1 to6, more preferably k=6;

b) a cyclic peptide of formula II:

(SEQ ID NO: 4) Cyclo(Ala-x-x-Trp-x-x-Gly-x-Phe-x-Cys-x_(h)-Gln), (II)whereby h is any integer from 0 to 2, preferably 1 or 2;

c) a cyclic peptide of formula III:

(SEQ ID NO: 5) Cyclo(Ala-x-x-x-x-x-x-x-x-x-Cys-x_(j)-Cys-x-x-x-Pro-x-Cys-Cys-x_(i)-Gln), (III)whereby j is any integer from 0 to 4, preferably j=4;whereby i is any integer from 0 to 6, preferably any integer from 1 to6, more preferably i=6; and

d) a peptide of formula IV:

(SEQ ID NO: 6)Ala-x_(l)-Cys-x_(m)-Cys-x-x-x-Pro-x-Cys-Cys-x_(n)-Gln,(IV)whereby 1 is any integer from 0 to 9, preferably any integer from 1 to9, more preferably n=9;whereby m is any integer from 0 to 4, preferably any integer from 1 to4, more preferably m=4;whereby n is any integer from 0 to 6, preferably any integer from 1 to6, more preferably n=6;whereby x is any amino acid, preferably any naturally occurring aminoacid, more preferably any naturally occurring L-amino acid.

In an embodiment the peptide is a cyclic peptide of formula Ia:

(SEQ ID NO: 7) cyclo(Ala-x₂-x-x₁-x-x₁-x₁-x-x₂-x₂-Cys-x-x-x₁-Pro-x-Cys-Cys-x_(k)-Gln), (Ia)whereby k is any integer from 0 to 6, preferably any integer from 1 to6, more preferably k=6;whereby x₁ is individually and independently selected from the groupcomprising acidic amino acids; andx₂ is individually and independently selected from the group comprisingbasic amino acids.

In an embodiment the peptide is a cyclic peptide of formula Ib:

(SEQ ID NO: 8) Cyclo(x₄-x₂-x₄-x₁-x₄-x₁-x₁-x₄-x₂-x₂-Cys-x₃-x₅-x₁-Pro-x₂-Cys-Cys-x₁-x₃-x₃-x₄-x₅-x₂-x₅),(Ib)whereby x₁ is individually and independently selected from the groupcomprising acidic amino acids;x₂ is individually and independently selected from the group comprisingbasic amino acids;x₃ is individually and independently selected from the group comprisingLeu, Ile, Val, Met, Trp, Tyr and Phe;x₄ is individually and independently selected from the group comprisingSer, Thr, Ala and Gly; andx₅ is individually and independently selected from the group comprisingGln, Asn

In an embodiment the peptide is a peptide of formula Ic:

(SEQ ID NO: 9) cyclo(Ala-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn- Arg-Gln). (Ic)

In a preferred embodiment at least one of acidic amino acid residues isreplaced by a different amino acid selected from the group comprisingacidic amino acids.

In a preferred embodiment at least one of the basic amino acid residuesis replaced by a different amino acid selected from the group comprisingbasic amino acids.

In a preferred embodiment at least one of the aliphatic amino acidresidues is replaced by a different amino acid selected from the groupcomprising aliphatic amino acids.

In a more preferred embodiment at least one Ala amino acid residue isreplaced by Glu.

In an embodiment the peptide is a cyclic peptide of formula IIa:

(SEQ ID NO: 10) Cyclo(Ala-x₄-x₂-Trp-x₁-x₃-Gly-x₄-Phe-x₃-Cys-x_(n)-Gln), (IIa)whereby n is any integer from 0 to 2, preferably 1 or 2;whereby x₁ is individually and independently selected from the groupcomprising acidic amino acids;x₂ is individually and independently selected from the group comprisingbasic amino acids;x₃ is individually and independently selected from the group comprisingLeu, Ile, Val, Met, Trp, Tyr and Phe; andx₄ is individually and independently selected from the group comprisingSer, Thr, Ala and Gly.

In a preferred embodiment the peptide is a peptide of formula IIb:

(SEQ ID NO: 11) cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-Gln), (IIb)or a peptide of formula IIc:

(SEQ ID NO: 12) cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Gln). (IIc)

In a more preferred embodiment at least one of the acidic amino acidresidues is replaced by a different amino acid selected from the groupcomprising acidic amino acids.

In another more preferred embodiment at least one of the basic aminoacid residues is replaced by a different amino acid selected from thegroup comprising basic amino acids.

In another more preferred embodiment at least one of the aliphatic aminoacid residues is replaced by a different amino acid selected from thegroup comprising aliphatic amino acids.

In another more preferred embodiment at least one Ala amino acid residueis replaced by Glu.

In preferred embodiment the peptide is a cyclic peptide of formula IIIa:

(SEQ ID NO: 13) Cyclo(Ala-x₄-x₂-x₃-x₁-x₃-x₃-x₄-x₃-x₃-Cys-x_(j)-Cys-x-x-x-Pro-x-Cys-Cys-x_(i)-Gln), (IIIa)whereby i is any integer from 0 to 6, preferably any integer from 1 to6, more preferably i=6;whereby j is any integer from 0 to 4, preferably any integer from 1 to4, more preferably j=4;whereby x₁ is individually and independently selected from the groupcomprising acidic amino acids;x₂ is individually and independently selected from the group comprisingbasic amino acids;x₃ is individually and independently selected from the group comprisingLeu, Ile, Val, Met, Trp, Tyr and Phe; andx₄ is individually and independently selected from the group comprisingSer, Thr, Ala and Gly.

In a more preferred embodiment the peptide is a peptide of formula IIIb:

(SEQ ID NO: 14) Cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gln), (IIIb)

In another more preferred embodiment at least one of the acidic aminoacid residues is replaced by a different amino acid selected from thegroup comprising acidic amino acids.

In another more preferred embodiment at least one of the basic aminoacid residues is replaced by a different amino acid selected from thegroup comprising basic amino acids.

In another more preferred embodiment at least one of the aliphatic aminoacid residues is replaced by a different amino acid selected from thegroup comprising aliphatic amino acids.

In another more preferred embodiment at least one Ala amino acid residueis replaced by Glu.

In a preferred embodiment the peptide is a linear peptide of formulaIVa:

(SEQ ID NO: 15)Ala-x₄-x₂-x₃-x₁-x₃-x₃-x₄-x₃-x₃-Cys-x_(m)-Cys-x-x-x-Pro-x-Cys-Cys-x_(n)-Gln, (IVa)whereby n is any integer from 0 to 6, preferably any integer from 1 to6, more preferably n=6;whereby m is any integer from 0 to 4, preferably any integer from 1 to4, more preferably m=4;whereby x₁ is selected from the group comprising acidic amino acids;

-   -   x₂ is selected from the group comprising basic amino acids;    -   x₃ is selected from the group comprising Leu, Ile, Val, Met,        Trp, Tyr and Phe; and    -   x₄ is selected from the group comprising Ser, Thr, Ala and Gly.

In a more preferred embodiment the peptide is a peptide of formula IVb:

(SEQ ID NO: 16) Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gln (IVb)

In a further aspect the present invention is related to cyclic peptideshaving any of the two following formulae, whereby it is to beacknowledged that both peptides are covered by formula IV but areadditional cyclic peptides:

(SEQ ID NO: 1) Cyclo(Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Gln) (SEQ ID NO: 2)Cyclo(Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Tyr-Gln)_.

In another more preferred embodiment at least one of the acidic aminoacid residues is replaced by a different amino acid selected from thegroup comprising acidic amino acids.

In another more preferred embodiment at least one of the basic aminoacid residues is replaced by a different amino acid selected from thegroup comprising basic amino acids.

In another more preferred embodiment at least one of the aliphatic aminoacid residues is replaced by a different amino acid selected from thegroup comprising aliphatic amino acids.

In another more preferred embodiment at least one Ala amino acid residueis replaced by Glu.

In an embodiment, in case the peptide is a cyclic peptide, thecyclization occurs by a linkage which is a covalent binding selectedfrom the group comprising S—S linkages, peptide bonds, carbon bonds suchas C—C or C═C, ester bonds, ether bonds, azo bonds, C—S—C linkages,C—N—C linkages and C═N—C linkages.

In a preferred embodiment the S—S linkage is formed by two Cys residuesof the peptide.

In an alternative preferred embodiment the peptide bond is formed by theNH₂ group of the N-terminal amino acid and the COOH group of theC-terminal amino acid.

In another alternative preferred embodiment additional bonds are formedby the side chain of NH₂ groups and COOH groups of the constituent aminoacids.

The problem underlying the present invention is solved in a secondaspect by a composition comprising at least one of the peptidesaccording to the first aspect of the present invention and a carrier.

The problem underlying the present invention is solved in a third aspectby a diagnostic agent comprising at least one of the peptides accordingto the first aspect of the present invention.

In an embodiment the diagnostic agent is for the detection ofanti-β-adrenergic receptor antibodies.

In a preferred embodiment the diagnostic agent comprises at least onefurther biologically active compound.

The problem underlying the present invention is solved in a fourthaspect by a diagnostic kit for the detection of anti-β1-adrenergicreceptor antibodies comprising a peptide according to the first aspectof the present invention or a diagnostic agent according to the thirdaspect of the present invention.

The problem underlying the present invention is solved in a fifth aspectby a pharmaceutical composition comprising at least one of the peptidesaccording to the first aspect of the present invention and apharmaceutically acceptable carrier.

In an embodiment the pharmaceutical composition additionally comprises afurther pharmaceutically active agent.

In a preferred embodiment the further pharmaceutically active agent isselected from the group comprising a beta receptor blocker, preferablyselective β1-adrenergic receptor blockers.

In a more preferred embodiment the further pharmaceutically active agentis selected from the group of selective β1-adrenergic receptor blockerscomprising atenolol, metoprolol, nebivolol, and bisoprolol or thenon-selective beta blocker carvedilol.

The problem underlying the present invention is solved in a sixth aspectby the use of a peptide according to the first aspect of the presentinvention, for the manufacture of a medicament.

In an embodiment the medicament is for the treatment and/or preventionof a disease, whereby such disease is selected from the group of heartdiseases, comprising infectious and non-infectious heart disease,ischemic and non-ischemic heart disease, inflammatory heart disease andmyocarditis, cardiac dilatation, idiopathic cardiomyopathy, idiopathicdilated cardiomyopathy, immune-cardiomyopathy, heart failure, and anycardiac arrhythmia including ventricular and supraventricular prematurecapture beats.

In a preferred embodiment the disease is idiopathic dilatedcardiomyopathy and preferably anti-β-AR antibody-induced dilatedimmune-cardiomyopathy.

In an embodiment the medicament comprises at least one furtherpharmaceutically active compound.

In an embodiment the medicament is for the treatment and/or preventionof patients having antibodies against β-adrenergic receptors, preferablyβ1-adrenergic receptors.

In an embodiment the medicament is for inducing immune tolerance.

The problem underlying the present invention is solved in a seventhaspect by the use of a peptide according to the first aspect of thepresent invention for the manufacture of a medicament, whereby themedicament is for inducing immune tolerance, preferably by suppressionof the production of anti-β-adrenergic receptor antibodies and morepreferably by suppression of the production of anti-β1-adrenergicreceptor antibodies.

The problem underlying the present invention is solved in an eighthaspect by the use of a peptide according to the first aspect of thepresent invention for inducing immune tolerance by suppression of theproduction of anti-β1-adrenergic receptor antibodies through blockade ofthe antigen-recognition sites of the antibody-producing prae B-cells.

In a still further aspect the problem underlying the present inventionis solved by the use of a peptide according to the present invention ina method of diagnosis.

In an embodiment the method is for the diagnosis of idiopathic dilatedcardiomyopathy, preferably anti-β-AR antibody-induced dilatedimmune-cardiomyopathy.

In preferred embodiment the method is a FRET-based method.

Without wishing to be bound by any theory in the following, the presentinventors have surprisingly found that a number of both cyclic as wellas linear peptides are, in principle, active as inhibitors ofanti-β-adrenergic receptor antibodies, more particularly as inhibitorsof anti-β1-adrenergic receptor antibodies. FIG. 2 demonstrates, however,that cyclic peptides are apparently two to three times better recognized(i.e. yield 2-3 times higher DO-values at a same antibody concentration)than linear peptides by anti-β-adrenergic receptor antibodies, moreparticularly by anti-β1-adrenergic receptor antibodies. In addition, themolecular architecture of a head-to-tail feature together withintramolecular cystein-bridges is thought to give the cyclic peptideshightened resitance to thermal, chemical, and enzymatic degradation invivo (Ireland D. C. et al. (2006) J. Mol. Biol. 12, 1-14).

It has to be acknowledged that, as also shown in FIG. 12 and FIG. 13,and described in examples 3 and 4, agents of the prior art, i.e. betablockers, which may be used for the treatment of dilated cardiomyopathyand other diseases which are caused by stimulatory anti-β1-adrenergicreceptor antibodies, such as bisoprolol, significantly reduced bothheart rate and blood pressure. In patients suffering from bronchialasthma which represents a contraindication for beta-blocking agentsbecause of a possible induction of bronchospasm or in patients sufferingalready from a low heart rate and/or a low systemic blood pressure it isthus not possible to use bisoprolol and similarly acting compounds,because a further decrease in heart rate or blood pressure might havesevere consequences, including death or the need for surgicalprocedures, i.e. the implantation of pace makers. In contrast thereto,the peptides of the present invention do not have a negative impact onlung function, heart rate or blood pressure and, therefore, are suitablefor the treatment of distinct patient groups which otherwise could notbe treated using a beta blocker, i.e. patients who already suffer frombradycardia for whom the use of beta blockers of the prior art such asbisoprolol, is not possible.

Also, the present inventors have surprisingly found that the peptidesaccording to the present invention obviously act through an additionalmechanism different from a capturing of the autoantibodies whichactivate the β1-adrenergic receptors resulting in, among others, dilatedimmune-cardiomyopathy and heart failure. Rather, the peptides accordingto the present invention seem to be suitable to induce immune tolerancesince endogeneous production of anti-β1-adrenergic receptor antibodiesrapidly decreased and finally stopped within 5-6 months in bothprophylactically (FIG. 4) and therapeutically (FIG. 8) treated animals.To the big surprise of the present inventors, within only a few months,significant immune responses were no more observed upon antigen boosts(given every 4 weeks) as shown in FIG. 4 and FIG. 8. This means thatsome kind of immunological tolerance comparable to hyposensitizationwith consecutive anergy to the β1-receptor antigen occurred, an effectmost likely due to a significant reduction and/or suppression, perhapseven apoptosis, of the anti-β1-EC_(II) producing B-cells. In fact,further analysis of the nature of this immunological tolerance revealeda significant decrease in antigen-specific antibody-producing splenicB-cells in the presence of the cyclic peptide (FIG. 20), whereasregulatory CD4⁺ T-cells and/or other mechanisms of T-cell inducedtolerance do not obviously account for this unresponsive state. FIG. 19shows representative T-cell recall-assays carried out with CD4⁺ T-cellsisolated from prophylactically (FIG. 19 a) or therapeutically (FIG. 19b) treated anti-β1-EC_(II) antibody-positive immunized animals. Theassay was performed according to the publication of Schmidt, J. et al.J. of Neuroimmunology 140, 143-152 (2003). Incubation of isolated CD4⁺T-cells with the cyclic peptide, especially the beta 1-ECII 25AA (aminoacid) peptide, named as the peptide of formula Ic, neither treatmentgroup resulted in a significant stimulation and subsequentlyproliferation of the T-cells compared with the dose-dependent cellproliferation reaction seen upon incubation of the T-cells with theβ1-ECII/GST fusion protein antigen (FP 1.0 μg/mL or 0.1 μg/mL), or theunspecific T-cell stimulator Concanavalin A (ConA, positive control). Incontrast, FIG. 20 shows the results from ELISPOT-assays with splenicB-cells demonstrating that the specifically anti-β1-ECII IgG secretingB-cells were markedly affected and significantly reduced in the spleensof animals treated with 1 mg/kg of the cyclic ECII-25AA peptide offormula Ic, whereas the everlasting anti-β1-ECII specific memory B-cellsin the bone marrow of treated animals were not affected by the cyclicpeptide. The non-β1-ECII-specific, that is total IgG producing B-cellsin the spleen or bone marrow necessary for any kind of humoral responseagainst foreign (including microbial) antigens were not affected at allin animals treated with 1 mg/kg of the cyclic peptide of formula Ic,excluding a general immuno-supressant effect of the cyclic peptide.

It will be understood that for the various peptides of the presentinvention, a certain flexibility and variability in the primarysequence, i.e. the amino acid sequence is possible as long as theoverall secondary and tertiary structure of the respective peptideswhich is defined by at least some fixed amino acid residues and by theirspatial arrangement, is ensured. Moreover the number of amino acids andthus the length of the primary structure appear to be crucial for thebiological effects of the various peptides of the present invention. Apeptide length equal to or above 26 amino acids (primary structure) isthought to be capable of stimulating directly (that is, without the useof carrier proteins) immunocompetent T-cells and thus may provoke aparadoxal increase in anti-β1-receptor antibody production throughT-cell mediated B-cell stimulation (data not shown). Therefore, in thesubsequent therapy study using a limited number of supplementary pilotanimals we tested shorter peptide variants of the 25 amino acidβ1-ECII-peptide, i.e. cyclic peptide-variants comprising either 18 or 16amino-acids (AA). The generated constructs were: ECII-18AA,cyclo(Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Gln)(SEQ ID NO:1); and ECII-16AA,cyclo(Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Tyr-Gln)(SEQ ID NO:2). Whereas the circulating anti-β1-EC_(II) antibody titerswere reduced by about a same extent with either the 25AA cyclic peptideIc or the 18AA construct (see FIG. 8 b)—both yielding also a similarbiological efficiency (i.e. reversal of the cardiomyopathic phenotype;see FIG. 10 d), the 16AA construct seems to be less effective withregard to both, the continuous reduction of circulating anti-β1-EC_(II)antibody titers (see grey diamonds, FIG. 8 b: upon peptide injection theantibody titers remain stable instead of decreasing) and biologicalefficiency (i.e. ambiguous results in 2 treated rats with one animalhaving complete reversal of the cardiomyopathic phenotype, the otheranimal progressive LV dilatation and dysfunction; see FIG. 10 d)indicating that a certain length of the cyclic receptor-homologouspeptides seems to be necessary to obtain the beneficial biologicaleffects.

The specific peptides in terms of defined amino acid sequences areparticularly preferred embodiments over the more general peptides, i.ethe peptides represented by the generic formulae herein.

In accordance therewith, the various generic formulae refer to a basicpeptide structure as reflected by formulae I, II, III and IV, wherebymore specific formulae and thus peptides being further defined in a lessgeneric manner but still being covered by formulae I, II, III and IVbeing referred to herein as Ia, Ib, Ic, IIa, IIb, IIIa, IIIb, IVa andIVb, respectively. It will also be understood by the one skilled in theart that the individual amino acid may be replaced by another naturallyoccurring or synthetic amino acid, preferably if both amino acids belongto the same category of amino acids. In accordance therewith, forexample, an acidic amino acid can be replaced by another acidic aminoacid, a basic amino acid may be replaced by another basic amino acid andso on. It will also be acknowledged by the ones skilled in the art thatone or several of the amino acids forming the peptide of the presentinvention may be modified. In accordance therewith any amino acid asused herein preferably also represents its modified form. For example,an alanine residue as used herein also comprises modified alanine. Suchmodifications may, among others, be a methylation or acylation or thelike, whereby such modification or modified amino acid is preferablycomprised by the present invention as long as the thus modified aminoacid and more particularly the peptide containing said thus modifiedamino acid is still functionally active as defined herein, moreparticularly functionally active as an inhibitor of β1-adrenergicreceptors and even more preferably active in inhibiting the interactionbetween β1-adrenergic receptors and antibodies, more preferablyauto-antibodies directed against β1-adrenergic receptors. Respectiveassays for determining whether such a peptide, i.e. a peptide comprisingone or several modified amino acids, fulfils this requirement, are knownto the one skilled in the art and, among others, also described herein,particularly in the example part hereof.

The invention comprises also derivatives of the peptides such as saltswith physiologic organic and anorganic acids like HCl, H₂SO₄, H₃PO₄,malic acid, fumaric acid, citronic acid, tatratic acid, and acetic acid.

As used herein, the sequences of the various peptides are indicated fromthe N-terminus to the C-terminus, whereby the N-terminus is at the leftside and the C-terminus is at the right side of the respective depictedamino acid sequence.

Preferably an acidic amino acid is an amino acid selected from the groupcomprising Asp, Asn, Glu, and Gln; preferably a basic amino acid is anamino acid selected from the group comprising Arg and Lys; preferably aneutral amino acid is an amino acid selected from the group comprisingGly, Ala, Ser, Thr, Val, Leu, Ile; preferably an aliphatic amino acid isan amino acid which is selected from the group comprising Gly, Ala, Ser,Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Arg, Lys, Cys and Met.

As used herein, the expression that one particular amino acid, such as,e.g., a basic amino acid, is replaced by a different amino acid which isselected from a respective particular group of amino acids, such as,e.g., the group comprising basic amino acids, preferably means that theparticular amino acid is replaced by another, i.e. different amino acidunder the proviso that such different amino acid is part of therespective particular group of amino acids. To the extent indicatedherein, this is applicable to each of the particular amino acid and, inprinciple, each such replacement is independent of any other replacementoptionally made in relation to other amino acids forming the respectivepeptide.

The peptides according to the present invention may be used as adiagnostic agent and for the manufacture of a medicament for thetreatment of diseases or may be used in a composition, preferably apharmaceutical composition, a diagnostic composition and a diagnostickit, preferably for the detection of anti-β-adrenergic receptorantibodies, more preferably for the detection of anti-β1-adrenergicreceptor antibodies. Such diseases are preferably those, where theβ1-adrenergic receptor is activated in a non-physiological manner, moreparticularly is activated by antibodies, more preferably byauto-antibodies which are directed against the β1-adrenergic receptor.More specifically, such diseases comprise, however, are not limitedthereto, the group of heart diseases, comprising infectious andnon-infectious heart disease, ischemic and non-ischemic heart disease,inflammatory heart disease and myocarditis, cardiac dilatation,idiopathic cardiomyopathy, idiopathic dilated cardiomyopathy,immune-cardiomyopathy, heart failure, and any cardiac arrhythmiaincluding ventricular and supraventricular premature capture. Suchpharmaceutical composition may additionally or alternatively also beused for the treatment of patients having antibodies againstβ-adrenergic receptors, preferably β1-adrenergic receptors. A furthersubgroup of patients which may be treated by the pharmaceuticalcomposition according to the present invention are those patientssuffering from any of the diseases described herein, more particularlythe group of heart diseases, comprising infectious and non-infectiousheart disease, ischemic and non-ischemic heart disease, inflammatoryheart disease and myocarditis, cardiac dilatation, idiopathiccardiomyopathy, idiopathic dilated cardiomyopathy,immune-cardiomyopathy, heart failure, and any cardiac arrhythmiaincluding ventricular and supraventricular premature capture and havingat the same time the antibodies directed against β-adrenergic receptors,more preferably antibodies against the β1-adrenergic receptor, wherebyin a preferred embodiment the antibodies are auto-antibodies. What issaid herein for the pharmaceutical composition applies also to themedicament for the manufacture of which the peptides of the presentinvention are used. As used herein, compounds and peptides are used inan interchangeable manner herein.

Apart from containing at least one peptide of the present invention, thecomposition may either comprise two or a plurality of cyclic peptides ofthe present invention and/or other β-receptor blockers, moreparticularly β1-adrenergic receptor blockers. Examples therefore are,among others, bisoprolol, metoprolol, atenolol, nebivolol, andcarvedilol. This kind of combination provides for protection fromantibody-induced selective β1-receptor-downregulation by the peptides(see FIGS. 17 and 18) going along with synergistic β1-receptorupregulation by beta-blockers like bisoprolol or metoprolol (see FIGS.17 and 18) and ultimately results in a synergistic effect as seen in theanimal model (see FIGS. 10, 11, and 13). In accordance therewith,reversal of the cardiomyopathic phenotype occurs only bycyclopeptide-monotherapy or by β-blocker/peptide combination-therapy,but not alone by betablocker monotherapy which has proven to bebeneficial in human heart failure and dilated cardiomyopathy, reportedin the CIBIS I, II and III studies and the MERIT-HF study.

The pharmaceutical composition typically comprises a carrier, morepreferably a pharmaceutically acceptable carrier, excipient or diluent.

For s.c. or i.v. injection, compounds of the invention may be formulatedin aqueous solution, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiologically salinebuffer. For transmucosal and transpulmonal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

The use of pharmaceutical acceptable carriers to formulate the compoundsaccording to the present invention into dosages or pharmaceuticalcompositions suitable for systemic, i.e. intravenous/intraarterial, orsubcutaneous administration is within the scope of the presentinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present invention, in particular thoseformulated as solutions, may be administered parenterally, such as byintravenous injection. The compounds can be readily formulated usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for subcutaneous or oral administration. Such carriers enablethe compounds according to the present invention to be formulated astablets, pills, capsules, dragees, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.

Compounds according to the present invention or medicaments comprisingthem, intended to be administered intracorporally/intracellularly may beadministered using techniques well known to those of ordinary skill inthe art. For example, such agents may be encapsulated into liposomes,then administered as described above. Liposomes are spherical lipidbilayers with aqueous interiors. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal microenvironment and, because liposomes fuse with cellmembranes, are efficiently delivered near the cell surface. Deliverysystems involving liposomes are disclosed in U.S. Pat. No. 4,880,635 toJanoff et al. The publications and patents provide useful descriptionsof techniques for liposome drug delivery and are incorporated byreference herein in their entirety.

Pharmaceutical compositions comprising a compound according to thepresent invention for parenteral and/or subcutaneous administrationinclude aqueous solutions of the active compound(s) in water-solubleform. Additionally, suspensions of the active compounds may be preparedas appropriate oily injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil or castor oil, orsynthetic fatty acid esters, such as ethyl oleate or triglycerides, orliposomes. Aqueous injections suspensions may contain compounds whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, or the like. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions and to allow for a constantly slow release of thesubstance in the organism.

Pharmaceutical compositions comprising a compound according to thepresent invention for oral use can be obtained by combining the activecompound(s) with solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, sorbitol, and the like; cellulosepreparations, such as, for example, maize starch wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidone (PVP) and the like, as well as mixtures of any twoor more thereof. If desired, disintegrating agents may be added, such ascross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereofsuch as sodium alginate, and the like.

Dragee cores as a pharmaceutical composition comprising a compoundaccording to the present invention are provided with suitable coatings.For this purpose, concentrated sugar solutions may be used, which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopolgel, polyethylene glycol, titanium dioxide, lacquer solutions, suitableorganic solvents or solvent mixtures, and the like. Preferably a gastricjuice resitent coating such as derivatives of cellulose Aquateric®,HP50® or HP55®, polymer of methacrylic acid and methacrylic acid esters(Eutragid® L, Eutragid® S; retard forms Eutragid® RL and Eutragid® RS orderivatives of polyvinyl are used. Dyestuffs or pigments may be added tothe tablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations comprising a compound according to thepresent invention which can be used orally include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

The pharmaceutical composition may be present in the range of 10 μg/kgto 100 mg/kg depending on the application form, preferably s.c. or i.v.application every two or four weeks. In the rat 1 mg/kg s.c. or i.v.every other month were sufficient to obtain therapeutic levels of thecompounds according to the present invention, with the respective dosagefor human preferably being about 1-10 mg/kg i.v. or s.c.

It is within the present invention that the pharmaceutical compositionis used for the treatment of any of the diseases and patient groups asdefined above including the detection of anti-β-receptor antibodies inthese patients by using the aforementioned compounds. Also, the peptidesaccording to the present invention may be used for the preparation of amedicament for the treatment and/or prevention of any of the diseasesand patient groups as defined above in connection with thepharmaceutical composition.

Finally, the present invention is related to a method for the treatmentof patients suffering from or being at risk to develop a disease asdisclosed herein, more particularly heart diseases, comprisinginfectious and non-infectious heart disease, ischemic and non-ischemicheart disease, inflammatory heart disease and myocarditis, cardiacdilatation, idiopathic cardiomyopathy, idiopathic dilatedcardiomyopathy, immune-cardiomyopathy, heart failure, and any cardiacarrhythmia including ventricular and supraventricular premature capturebeats, whereby the patient is in need of such treatment and whereby themethod comprises administering to said patient a pharmaceuticallyeffective amount of the peptide of the present invention, or thepharmaceutical composition or the medicament disclosed herein.Preferably, a therapeutically effective dose refers to that amount ofthe active ingredient that produces amelioration of symptoms or aprolongation of survival of a subject which can be determined by the oneskilled in the art doing routine testing.

A “patient” for the purposes of the present invention, i.e. to whom acompound according to the present invention or a pharmaceuticalcomposition according to the present invention is administered, includesboth humans and other animals and organisms. Thus the compounds,pharmaceutical compositions and methods are applicable to or inconnection with both human therapy and veterinary applications includingdiagnostic(s), diagnostic procedures and methods as well as stagingprocedures and methods. For example, the veterinary applicationsinclude, but are not limited to, canine, bovine, feline, porcine,caprine, equine, and ovine animals, as well as other domesticatedanimals including reptiles, such as iguanas, turtles and snakes, birdssuch as finches and members of the parrot family, lagomorphs such asrabbits, rodents such as rats, mice, guinea pigs and hamsters,amphibians, fish, and arthropods. Valuable non-domesticated animals,such as zoo animals, may also be treated. In the preferred embodimentthe patient is a mammal, and in the most preferred embodiment thepatient is human.

In a further aspect, the present invention is related to a method fordiagnosing a patient which can be treated using the peptide,pharmaceutical composition and medicament according to the presentinvention. Preferably, such method comprises the steps as described inthe example part hereof. In one aspect, the present invention is thusrelated to the use of peptides according to the present invention as adiagnostic or for the manufacture of a diagnostic test. The rationalebehind this diagnostic use of the peptides according to the presentinvention is their interaction with the structures described above andin particular with the anti-β-adrenergic receptor antibodies.

So far, the definition of antibody-positivity depends on highlydivergent screening methods such as, e.g., ELISA with receptor peptides,Western blotting of heart tissues, functional assays with neonatal ratcardiomyocytes, or detection by surface plasmon resonance. Until now,this issue has not been solved satisfactorily.

One approach for using the peptides according to the present inventionas a diagnostic and in a diagnostic method, respectively, is athree-step screening procedure comprising performing an ELISA with thepeptides according to the present invention as well as determiningimmunofluorescence and determining cAMP responses in cells expressingnative human beta-AR. It is to be acknowledged that each and any of theaforementioned steps can as such be preformed for the detection of saidantibodies using the peptides according to the present invention. Alarge number of heart failure patients may thus be screened forfunctionally active anti-beta1-Abs. In connection with such diagnosticmethod the definition of functionally active anti-beta1-Abs ispreferably based on their effects on receptor-mediated signalling, thatis, their effects on cellular cAMP levels and on the activity of thecAMP-dependent protein kinase (PKA). Cyclic AMP is an universal secondmessenger of many G protein-coupled receptors including thebeta-adrenergic receptor family. It exerts its effects via PKA,cAMP-gated ion channels, phosphodiesterases, and exchange proteinsdirectly activated by cAMP, known as Epac1 and 2. The prior artdescribes several fluorescence methods for measuring cAMP in intactcells which can all be used in connection with the diagnostic method ofthe present invention. Fluorescence resonance energy transfer (FRET)between green fluorescent protein (GFP) variants fused to the regulatoryand catalytic subunits of PKA has been described to study thespatio-temporal dynamics of cAMP in neurons (Hempel C M, Vincent P,Adams S R, Tsien R Y, Selverston A I. Nature 1996, 384:113-114) orcardiac myocytes (Zaccolo M, Pozzan T., Science 2002, 295:1711-1715).

More recently, single chain fluorescence indicators have been describedin the art which are characterized by having an enhanced cyan (CFP) oryellow fluorescent protein (YFP) directly fused to the cAMP-bindingdomain of Epac-proteins, which allowed to achieve a higher sensitivityand better temporal resolution of the cAMP measurements. Such system is,among others described in WO2005/052186 the disclosure of which isincorporated herein by reference in its entirety. Such system can beused in connection with any diagnostic procedure using the peptidesaccording to the present invention. Also such system can be used for,however is not limited thereto, analyzing the prevalence of functionallyactive anti-beta1-Abs. Preferably such diagnostic method is applied to acohort of previously antibody-typed DCM patients or any individual to beassessed insofar or any individual suspected of suffering from any ofthe diseases described herein or being at risk to suffer therefrom. In afurther step of the diagnostic method and screening method, the abilityof beta-blockers to inhibit anti-beta1-Ab-induced receptor activationcan be assessed and determined, respectively.

The afore described assay which is a FRET-based method as described inWO 2005/052186 making use of the peptides according to the presentinvention is advantageous insofar as it is simpler, less time consuming,and at the same time discloses or identifies all DCM patients previouslyconsidered anti-beta1-ECII-positive. This embodiment of a FRET basedmethod of diagnosing making use of one or several of the peptidesaccording to the present invention is based on detectingantibody-induced increases in cAMP. This second messenger most likelyaccounts for the harmful effects provoked by activating anti-beta1-Abs.Therefore, it is advantageous that this method, which is preferably usedas a diagnostic method, and which quantifies antibody-induced cAMPsignals allows to differentiate between functionalanti-beta1-Ab-classes. In fact, by applying this method a previouslyundetected patient population characterized by the presence of “low”activating autoantibodies presumably targeting a different receptordomain, has been identified. Additionally, this method suggests thatthese classes are preferably directed against different epitopes of thereceptor. Taken together, screening by Epac-FRET appears to represent avery sensitive single step approach, allowing to detect different kindsof activating antibodies directed against the human beta1-AR. Therefore,the present invention is also related to the use of one or several ofthe peptides according to the present invention for use in an Epac-FRETassay. More preferably such Epac-FRET assay is used for diagnosis, evenmore preferably for the diagnosis of patients suffering from orsuspected of suffering from any of the disease described herein.

A further finding underlying the present invention is that functionalanti-beta 1-Abs were detected in almost two thirds of patients withDCM—about half of them “high” activator IgG, it means directed againstbeta 1-ECII, and about another half “low” activator IgG, it meansdirected against beta 1-ECI. Insofar, these patients define groups ofpatients which can be diagnosed and treated, respectively, with thepeptides according to the present invention. Mortality curves for thetwo FRET-classified populations demonstrated that “low” activator IgGpatients do not significantly differ in mortality from DCM patientswithout activating auto-antibodies, whereas “high” activator IgGpatients have a significantly higher mortality. Thus, anti-beta 1-Absdirected against beta 1-ECII are clinically particularly relevant andare, therefore, a particularly preferred marker and target,respectively, in or subject to the diagnostic methods disclosed herein.The capability of the FRET-based approach which is a preferred method ofdiagnosis according to the present invention to differentiate betweenprognostically distinct anti-beta1-Abs in heart failure, preferably dueto DCM, provides for the clinical relevance of this method.

In a still further aspect the present invention is related to adiagnostic agent. Such diagnostic agent consists of or comprises atleast one peptide of the present invention. Preferably the diagnosticagent consists of a peptide of the present invention, whereby thepeptide preferably comprises a label. Such label may be selected fromthe group comprising radioactive labels and fluorescent labels.Respective labels are known to the ones skilled in the art. Typically,the peptide is the part of the diagnostic agent conferring specificbinding characteristics to the diagnostic agent, preferably binding toanti-β1-adrenergic receptor antibodies, whereas the label confers thesignalling characteristics to the diagnostic agent.

The diagnostic agent may comprise, apart from the labelled or unlabelledpeptide of the present invention, a further biologically activecompound. Such further biologically active compound, in a preferredembodiment, is a means to confer signalling characteristics to thediagnostic agent, particularly in case the peptide of the presentinvention is unlabelled. For example, the further biologically activecompound can be an antibody, preferably a monoclonal antibody, and morepreferably a labelled antibody specifically binding to a peptide of thepresent invention or to a complex consisting of a peptide of the presentinvention and an anti-β-adrenergic receptor antibody, preferably ananti-β1-adrenergic receptor antibody.

The kit in accordance with the present invention comprises at least afeature which is selected from the group comprising a peptide of thepresent invention and a diagnostic agent according to the presentinvention. In an embodiment the kit further comprises an instructionleaflet, and/or a buffer for use in the application of the kit, and/orat least one reaction vessel for carrying out the detection reaction forwhich the kit is or is to be used. In a further embodiment, some or allof the reagents used in connection with the application of said kit arepresent as portions useful in carrying out the reaction(s) for which thekit is to be used.

In preferred embodiments, the following abbreviations shall have thefollowing meanings: Ab or ab: antibody, Abs or abs: antibodies, AR:adrenergic receptor, EC extracellular and AA amino acid.

The present invention will now be further illustrated by the followingfigures and examples, from which further advantages, features andembodiments may be taken, whereby.

FIG. 1 shows different therapeutic approaches for the treatment ofautoantibody-induced dilated immune-cardiomyopathy. The beta-adrenergicreceptor-mediated signaling cascade (depicted are the beta-receptor(beta-AR), the G protein (subunits Gs-alpha and -beta, gamma, and theadenylatcyclase (AC)) and their blockade by a beta blocking agent (leftpanel) or a cyclic peptide (right panel) is demonstrated. Onetherapeutic mode of action of the peptide is illustrated, that iscapturing of the antibodies against the beta1-adrenergic receptor by thecyclic peptide.

FIG. 2 demonstrates the difference in recognition of the linear versusthe cyclic 25AA beta 1-ECII peptide, namely peptide Ic, by specificanti-beta1-ECII receptor-antibodies generated in CrlBR Lewis rats usingbeta1-ECII/GST as antigen. Columns represent the amount of specificanti-beta 1-ECII antibodies bound to either linear (black) or cyclic(white) beta 1-ECII-peptides. Representative results obtained from sixdifferent rats are shown.

FIG. 3 is a schematic representation of the various experimental set upsfor the prevention and/or therapy of immune cardiomyopathy, whereby“immune cardiomyopathy” means immunized non-treated animals(cardiomyopathic phenotype); “bisoprolol-prevention/-therapy” meansimmunized animals treated in a prophylactic or therapeutic manner usingbisoprolol; and “peptide-prevention/-therapy” means immunized animalstreated using a peptide according to the present invention; “add-on”therapy means parallel treatment of immunized animals with bisoprolol(15 mg/kg, administered orally) and the cyclic peptide Ic (1 mg/kg,intravenously). Lewis CrlBR rats were used as animals. The numbers ofanimals used in the various experimental branches are indicated and theend of each treatment arm; data shown in all following figures refer tothese numbers; bisoprolol controls were treated with 15 mg/kgadministered orally, peptide controls with 1 mg/kg i.v. or s.c. andNaCl/GST controls s.c.

FIG. 4 is a diagram indicating the titer course of specific anti-1-ECIIantibodies in the prevention arm of the study, whereby “Beta1 untreated”means immunized animals being not treated, “Beta1/bisop.” meansimmunized animals being prophylactically treated using bisoprolol in adosage of 15 mg/kg, and “Beta1/pept. ECII-25AA i.v.” and “Beta1/pept.ECII-25AA s.c.” means immunized animals being prophylactically treatedintravenously (i.v.) or subcutaneously (s.c.) using the peptide Ic indosage of 1 mg/kg each.

FIG. 5 is the time course of the heart rate of animals in the preventionarm of the study, whereby “Beta1 untreated” means immunized not treatedanimals, “Beta1/bisop.” means immunized animals prophylactically treatedwith bisoprolol, the first 4 months 10 mg/kg then 15 mg/kg, “Beta1/peptECII-25AA” means immunized animals prophylactically treated with thepeptide Ic (1 mg/kg; n=4 subcutanously, n=4 intravenously); and “Cont.”means corresponding control groups; “bpm” means beats per minute.

FIG. 6 is a diagram showing the internal end-systolic and end-diastolicleft ventricular diameters as determined by echocardiography(echocardiographic system: Vivid Seven, GE Vingmed Ultrasound, Horten,Norway, equipped with a 10-12.5 MHz transducer), whereby “Beta1untreated” means immunized not treated animals, “Beta1/bisop.” meansimmunized animals prophylactically treated with bisoprolol, “Beta1/pept.ECII-25AA” means immunized animals prophylactically treated with the 25AA peptide Ic; and “Cont.” means corresponding control groups, wherebyLVES/LVED is left ventricular end-systolic diameter/left ventricularend-diastolic diameter.

FIG. 7 is a diagram showing the “Cardiac index” in ml/min/g (bodyweight) as determined by echocardiography (echocardiographic system seeabove), whereby “Beta1 untreated” means immunized not treated animals,“Beta1/bisop.” means immunized animals prophylactically treated withbisoprolol, “Beta1/pept.” means immunized animals prophylacticallytreated with the beta 1-ECII 25AA peptide Ic; and “Cont.” meanscorresponding control groups.

FIG. 8 a is a diagram indicating the titer course of specific anti-ECIIantibodies in the therapy arm of the study, given as % of the respectivetiters at the time point treatment was initiated (“starting value”),whereby “Beta1 untreated” means immunized anti-1-positivecardiomyopathic not treated animals, “Beta1/bisop.” means immunizedanti-1-positive cardiomyopathic animals treated with bisoprolol (15mg/kg, orally), “Beta1/pept. ECII-25AA” means immunized anti-1-positivecardiomyopathic animals treated with the 25AA peptide Ic in a dosage of1 mg/kg intravenously, and “Beta1/bisop.+pept.25AA” means treatment witha combination of bisoprolol and the 25AA peptide Ic. Therapy was startedafter 8 months of regular (1× monthly) immunization. Monthlyimmunization was continued under therapy.

FIG. 8 b is a diagram comparing the titer course of specific anti-ECIIantibodies dependent on the different peptide variants used in thetherapy arm of the study, whereby 25AA means the cyclic peptide Ic, 18AAmeans the shorter 18 amino-acid variant, and 16AA the shorter 16amino-acid variant of the peptide. “Beta1 untreated” means immunizedanti-1-positive cardiomyopathic not treated animals, “Beta1/pept. ECI”means immunized anti-1-positive cardiomyopathic animals treated with apeptide corresponding to the first extracellular receptor loop (1 mg/kg,intravenously, negative control—supposed and shown to not affect theanti-ECII antibody-titers), “Beta1/pept. ECII-25AA, ECII-18AA,ECII-16AA” means immunized anti-1-positive cardiomyopathic animalstreated with the 25AA-, 18AA-, or 16AA-peptide, respectively.

FIG. 9 is a diagram indicating the heart rate of the animals from thetherapy study, whereby “Beta1 untreated” means immunized anti-1-positivecardiomyopathic not treated animals; “Beta1/bisop.” means immunizedanti-1-positive immunized cardiomyopathic animals treated withbisoprolol (15 mg/kg) after 8 months; “Beta1/pept ECII-25AA, -18AA,-16AA” means immunized anti-1-positive cardiomyopathic animals treatedwith 1 mg/kg of the 25AA-, 18AA-, or 16AA-peptide, respectively, after 8months of immunization, and “Cont.” means the respective control groups;“Beta1/bisop.+Pept ECII-25AA” means immunized anti-1-positivecardiomyopathic animals treated with a combination of bisoprolol and thebeta 1-ECII 25AA peptide after 8 months.

FIG. 10 is partitioned in 4 diagrams, each showing the time course ofinternal end-systolic and end-diastolic left ventricular diameters asdetermined by echocardiography, whereby “Beta1 untreated” meansimmunized anti-1-positive cardiomyopathic not treated, and “Cont.” notimmunized control animals plotted together with (a) immunizedanti-1-positive cardiomyopathic animals treated with 15 mg/kg bisoprolol(“Beta1/bisop.”), not immunized control animals treated with bisoprolol(“Bisop. cont.”), (b) immunized anti-1-positive cardiomyopathic animalstreated with 25AA cyclic peptides, namely peptide Ic, (1 mg/kg, starting8 months after immunization; “Beta1/pept ECII-25AA”), and not immunizedcontrol animals injected with the same peptide (“Pept. cont.”), (c)immunized anti-1-positive cardiomyopathic animals treated withbisoprolol (“Beta1/bisop.”), 25AA cyclic peptides (“Beta1/peptECII-25AA”), or a combination of bisoprolol/cyclic peptide(Beta1/bisop.+Pept. ECII-25AA), and (d) immunized anti-1-positivecardiomyopathic animals treated with ECII-25AA/18AA/16AA peptides, orthe ECI-peptide, namely petide IIb or IIc, respectively (“Beta1/peptECII-25AA, ECII-18AA, ECII-16AA”, or “Beta1/pept ECI”). LVES means leftventricular end-systolic diameter and LVED means left ventricularend-diastolic diameter.

FIG. 11 is partitioned in 4 diagrams depicting the time course of the“Cardiac index” in ml/min/g (body weight) as determined byechocardiography (echocardiographic system see above), whereby “Beta1untreated” means immunized anti-1-positive cardiomyopathic not treated,and “Cont.” not immunized control animals plotted together with (a)anti-1-positive immunized animals treated with 15 mg/kg bisoprolol(“Beta1/bisop.”), not immunized control animals treated with bisoprolol(“Bisop. cont.”), (b) immunized anti-1-positive cardiomyopathic animalstreated with 25AA cyclic peptides, namely peptide Ic, (1 mg/kg, starting8 months after immunization; “Beta1/pept ECII-25AA”), and not immunizedcontrol animals injected with 25AA cyclic peptides (“Pept. cont.”), (c)immunized anti-1-positive cardiomyopathic animals treated withbisoprolol (“Beta1/bisop.”), 25AA cyclic peptides (“Beta1/peptECII-25AA”), or a combination of bisoprolol/cyclic peptide(Beta1/bisop.+Pep. ECII-25AA) after 8 months, and (d) immunizedanti-1-positive cardiomyopathic animals treated with ECII-25AA/18AA/16AApeptides, or the beta 1-ECI-peptide preferably peptide IIb and IIc,respectively (“Beta1/pept ECII-25AA, ECII-18AA, ECII-16AA”, or“Beta1/pept ECI” (negative control)).

FIG. 12 shows hemodynamic parameters obtained in the prevention study,in detail the heart frequency (a), the LV systolic blood pressure (b),the LV end-diastolic pressure (c) and contractility/relaxation (d),whereby “Beta1 untreated” means immunized not treated animals,“Beta1/bisop.” means immunized animals prophylactically treated withbisoprolol, “Beta1/pept ECII-25AA” means immunized animalsprophylactically treated with the cyclic beta 1-ECII 25AA peptide Ic and“Cont.” means respective control groups.

FIG. 13 shows hemodynamic parameters obtained in the therapy study, indetail the heart frequency (a), the LV systolic blood pressure (b), theLV end-diastolic pressure (c) and contractility/relaxation (d), whereby“Beta1 untreated” means immunized anti-beta1-positive cardiomyopathicnot treated animals, “Beta1/bisop.” means immunized anti-1-positivecardiomyopathic animals therapeutically treated with bisoprolol,“Beta1/pept ECII-25AA” means immunized anti-1-positive cardiomyopathicanimals therapeutically treated with the cyclic beta1-ECII 25AA peptideIc, or with a combination of bisoprolol/cyclic peptide (“Add-on”) after8 months of immunization, and “Cont.” means the respective controlgroups.

FIG. 14 shows different laboratory parameters determined in the serum ofanimals at the end the prevention (upper panels) and the therapy study(lower panels), respectively, whereby “Beta1 untreated” means immunizedanti-1-positive not treated animals, “Beta1/bisop.” means immunizedanimals prophylactically (upper panels) or therapeutically (lowerpanels) treated with bisoprolol, “Beta1/pept.” means immunized animalsprophylactically (upper panels) or therapeutically (lower panels)treated with the 25AA-peptide Ic, “Add-on” means immunizedanti-1-positive animals therapeutically treated with a combination ofbisoprolol and the cyclic 25AA-peptide Ic after 8 months ofimmunization, and “Cont.” means the respective control groups in boththe prevention and the therapy study. Crea means creatinine, Hst-N meansurea, GOT means glutamic oxaloacetic transaminase, Bili means bilirubin,GLDH means glutamate lactate dehydrogenase.

FIG. 15 shows macro anatomic parameters of the animals from theprevention study as columns. The relative weights of different organswere determined in g, whereby “Beta 1 untreated” means immunized animalsbeing not treated, “Beta1/bisop.” means immunized animalsprophylactically treated with bisoprolol in a dosage of 15 mg/kg and“Beta1/pept. ECII-25AA” means immunized animals prophylactically treatedusing the 25AA-ECII peptide Ic in dosage of 1 mg/kg each; “Cont.” meansthe respective control groups; Kidney R means right and Kidney L meansleft.

FIG. 16 shows macro anatomic parameters of the animals from the therapystudy as columns. The relative weights of different organs weredetermined in g, whereby “Beta1 untreated” means immunizedanti-1-positive cardiomyopathic animals being not treated,“Beta1/bisop.” means immunized anti-1-positive cardiomyopathic animalstherapeutically treated with bisoprolol in a dosage of 15 mg/kg,“Beta1/pept. ECII-25AA” means immunized anti-1-positive cardiomyopathicanimals therapeutically treated with the 25AA-ECII peptide Ic in dosageof 1 mg/kg each, “Add-on” means therapeutic treatment of immunizedanti-1-positive cardiomyopathic animals with a combination ofbisoprolol/cyclic peptide after 8 months of immunization, and “Cont.”means the respective control groups. Kidney R means right and Kidney Lmeans left.

FIG. 17 shows densities of cardiac beta-adrenergic receptors in theheart of the animals from the prevention study as columns. The upperpanel shows the total amount of cardiac membrane beta-AR, given infemtomol per milligram (fmol/mg) membrane protein. The lower panel showsthe amount of the beta2-AR (left) and beta1-AR subtypes (right),respectively. “Beta1 untreated” means immunized anti-1-positive animalsbeing not treated, “Beta1/bisop.” means immunized anti-1-positiveanimals prophylactically treated with bisoprolol in a dosage of 15 mg/kgand “Beta1/pept. ECII-25AA” means immunized anti-1-positive animalsprophylactically treated using the 25AA-ECII peptide Ic in dosage of 1mg/kg each, “Cont.” means the respective control groups.

FIG. 18 shows densities of cardiac beta-adrenergic receptors in theheart of the animals from the therapy study as columns. The upper panelshows the total amount of cardiac membrane beta-AR, given in femtomolper milligram (fmol/mg) membrane protein. The lower panel shows theamount of the beta2-AR (left) and beta1-AR subtypes (right),respectively. “Beta1 untreated” means immunized anti-1-positivecardiomyopathic animals being not treated, “Beta1/bisop.” meansimmunized anti-1-positive cardiomyopathic animals therapeuticallytreated with bisoprolol in a dosage of 15 mg/kg and “Beta1/pept.ECII-25AA” means immunized anti-1-positive cardiomyopathic animalstherapeutically treated using the 25AA-ECII peptide Ic in dosage of 1mg/kg each; “Add-on” means therapeutic treatment of immunizedanti-1-positive cardiomyopathic animals with a combination ofbisoprolol/cyclic peptide after 8 months of immunization, and “Cont.”means the respective control groups.

FIG. 19 shows the results of T-cell recall-assays carried out withT-cells prepared from the spleen of immunized anti-1-positivecardiomyopathic not treated animals (“Beta1 untreated”) compared withthose from immunized anti-1-positive cardiomyopathic animals (a)prophylactically or (b) therapeutically treated with bisoprolol(Beta1/bisop.) or the cyclic 25AA-ECII cyclopeptide Ic (“Beta1/pept.ECII-25AA”). “Cont.” means the respective not immunized control animals.The assays were carried out according to Schmidt J. et al. (2003) JNeuroimmunol. 140, 143-152. In brief, to purify CD4+ T-cells from thesplenic cell preparations, B-cells and CD8+ T-cells were depleted bycommercially available magnetic beads (MACS®), yielding a purity of CD4+cells >85%. 1×10⁵ CD4+ of the purified T-cells were then co-incubated in96-well plates with 1×106 irradiated thymic antigen presenting cells(prepared from a younger rat). Reagents added in the different assays(conditions) were: 10 μg/ml glutathion-S-transferase (GST), 2 μg/mlConcanavalin A (ConA, positive control), 10 μg/ml 25AA-ECII peptide(Pept.25AA), and GST/beta1-ECII fusion protein (FP) at a concentrationof 1.0 and 0.1 μg/ml, respectively. The measured T-cell reactivitieswere normalized to the respective T-cell reactivities obtained with 10μg/ml purified protein derivative (PPD) carried out in parallel undereach condition. After 48 hours of incubation, 50% of the supernatantswere removed and replaced by fresh medium. The cells were then pulsedwith 1.25 μCi/well [³H]-thymidine and further incubated for 16 hoursbefore the cells were harvested, and the DNA-incorporated radioactivitywas measured using a beta-plate.

FIG. 20 shows the results of ELISPOT-assays carried out with B-cellsprepared from either the spleen or the bone marrow of immunizedanti-1-positive cardiomyopathic not treated animals (“Beta1 untreated”)compared with those isolated from immunized anti-1-positivecardiomyopathic animals therapeutically treated with the 25AA-ECIIpeptide Ic (“Beta1/pept. ECII-25AA”). For the assays, ELIspot plateswere coated overnight with either 1.8 μg/ml anti-rat IgG (H+L) or thespecific antigen (GST/beta1-ECII-FP) in 0.05 mol/l Tris buffer, pH 9.4.Then the plates were washed 3 times and blocked with BSA for 3 hours at37° C. Subsequently, the plates were incubated overnight at 37° C. withB-cells from either spleen or bone marrow (cultured in RPMI1640/X-VIVO-15 medium supplemented with 10% fetal calf serum (FCS)) with1×106 to 1×103 cells per well. After 12 hours the B-cells were discardedand the plates with the B-cell secreted IgG bound were washed severaltimes (PBS/0.5% Tween) before the addition of alkaline phosphataseconjugated secondary anti-rat IgG (0.3 μg/ml) to detect bound rat IgG.Then the plates were incubated for another 3 hours at 37° C., washedseveral times with PBS/0.5% Tween, and developed using LMP/BICP 5:1 (1ml per well; “LMP” means low melting agarose, and “BICP” means5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, a blue-coloreddye) allowing for a quantification of the blue spots obtained, with eachspot representing either (a) an IgG or (b) an antigen-specific IgGsecreting spleen or bone-marrow cell, respectively. Panel a depicts thetotal amount of IgG secreting cells per 104 coated cells, panel bdepicts the fraction of specifically anti-beta1-ECII IgG secretingcells; “BM” means bone marrow.

FIG. 21 represents an example of the spectroscopic characterization ofthe 25AA-ECII, namley the cyclic peptide Ic by high pressure liquidchromatography (HPLC, FIG. 21), and mass spectroscopy method (MALDI-TOF,data not shown). HPLC was carried out in a Waters Separation Modul 2690together with a Waters Dual Lambda UV detector, wave length set at 220nm. After peptide-synthesis and cyclization, the samples were dissolvedin H2O/5% acetonitril (ACN) and loaded on a reversed phase column(Nuclosil 100-5/C18, Macherey-Nagel Inc., Germany; column length 250 mm,diameter 4 mm) with a flow of 1 ml/min. For separation a gradient 5% to60% ACN with 0.2% TFA was used. A very small peak of linear 1-ECII-25AApeptide was seen, typically between 14 and 16 min, whereas the fractionscontaining the cyclic beta1-ECII-25AA peptide appeared in a range from18 to 22 min. Aliquots of these fractions containing 20-80 μg/ml of thecyclic peptide were further analyzed by mass spectroscopy. The expectedmolecular masses of the linear versus cyclic beta1-ECII-25AA are2948-2949 versus 2929-2930, respectively. The example demonstrates arepresentative mass spectrum of a cyclic beta1-ECII 25AA peptide. Theordinate demonstrates signal intensities (“a.u.” means arbitrary units),the abscissa, and the molecular mass (m/z) (data not shown).

FIG. 22 shows a cell-based approach for the detection of functionalanti-beta1-Abs. a, Receptor activation induced by binding of antibodiesto its accessible extracellular loops leads to an increase in cAMPthrough sequential activation of Gs-proteins and adenylyl cyclase (AC)which is detected by FRET as a conformational change in the cAMP-bindingdomain of Epac1 fused between CFP (cyan) and YFP (yellow) proteins(Epac1-camps). b, Measuring cAMP levels (fmol/cell) inisoproterenol-stimulated HEKβ1AR cells by conventional radio-immunoassay(Amersham) or c, Epac-FRET. One of 3 independent experiments(EC50=0.53±0.19 nM) is shown. The cAMP-range that can be monitored byEpac-FRET is presented by the horizontal grey bar in FIG. 22 b. At 0.1nM isoproterenol Epac1-camps gets saturated indicating an intracellularcAMP concentration at ˜20 μM. This extremely sensitive sensor ischaracterized by a high dynamic range at physiologically relevant cAMPconcentrations 0.1-20 μM (which are covered by only 10% of the maximalcAMP-RIA signal), making minor, in conventional assays not-significantcAMP changes well detectable by Epac-FRET. d, IgG prepared from ratsimmunized with the second extracellular loop of the human beta1-AR(beta1-ECII) were tested for activity using HEKβ1AR cells transfectedwith Epac1-camps, and compared with non-immunized animals to assess thereliability of the method. FRET ratio traces are presented (%corresponds to the relative change in YFP/CFP intensity ratio; Iso,(−)Isoproterenol). The decrease in FRET reflects a rise in intracellularcAMP (representative experiments, n=6).

FIG. 23 shows the measurement of cAMP by Epac-FRET detects anti-beta1-Abs in DCM patients. a, None of the IgG prepared from healthy controls(n=40) induced a significant cAMP-response in living cells (left). IgGfrom DCM patients previously judged anti-beta1-Ab-positive 5 (Abs+)elicited marked cAMP responses (49.5±3.8% of maximal Iso-signal,middle). IgG from 33.9% of previously anti-beta 1-Ab-negative judgedpatients (Abs-) demonstrated a robust but significantly smaller increasein cAMP (30.7±5.6%, P<0.01). Representative experiments of at least 3cells for each serum. b, Histogram with normality curves of the strengthof the FRET signal in healthy controls and DCM patients. Theantibody-induced FRET responses allow to differentiate between controlsand three groups of activity: no activity (n=17), low activity (n=19),high activity (n=20); P<0.01 for the difference between “high” and“low”-activators). c, Concentration-response relation between “high” and“low” activator IgG demonstrating different activatory capacities in awide range of antibody concentrations. The % ±SEM of maximal Iso-inducedcAMP response (normalized to maximal changes in FRET produced by a“high” activator) is presented (representative experiments, n=4).

FIG. 24 shows blocking experiments for different classes ofanti-beta1-Abs. a, cAMP production induced by a “high” activator isattenuated only by a specific peptide derived form the secondextracellular beta1-AR loop (beta 1-ECII). b, “Low” activator signalscould be blocked in all instances by a peptide corresponding to thefirst extracellular loop (beta1-ECI) but not by beta1-ECII-peptides(representative experiments from at least 3 cells per condition). c,Results of the cross-blocking experiments with IgG obtained from n=7representative “high”, and n=8 representative “low” activator DCMpatients.

EXAMPLE 1 Peptide Synthesis

Linear peptides are synthesised using the solid phase Fmoc protocol on aMultiple Peptide Synthesizer (SYROII, MultiSynTech GmbH, Witten). Thesynthesis was performed using side chain protected Fmoc amino acidderivatives on Rink Amide or Rink Amide MBHA resins (Novabiochem-MerkBiosciences GmbH, Bad Soden). The Fmoc amino acids are activated throughdiisopropylcarbodiimide/N-hydroxybenzotriazole orbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate.

For the synthesis of cyclic peptides on the solid phase Fmoc-Glu-ODmabor another Fmoc amino acid having a side chain protecting group whichcan be selectively cleaved off in an orthogonal manner, is incorporatedat the C-terminal end of the linear peptide. After selective removal ofthe Dmab side chain using 2% hydrazine monohydrate inN,N′-dimethylformamide the resin-bound linear peptide is treated in thepresence of diisopropylcarbodiimide and N-hydroxy-9-azabenzotriazole inN,N′-dimethylformamide for several hours. The cyclisation was monitoredby taking samples and performing the Kaiser-Test and, if necessary,repeated. The cleaving off of the cyclic peptide from the synthesisresin generates a peptide amide and the removal of the protective groupsof the side chain is done by treating the resin with trifluoro aceticacid/triisopropylsilane/ethandithiole/water for 2 hours.

EXAMPLE 2 Animal Model

The animal model used in this example and any other example describedherein if not indicated to the contrary, is the human analogue ratmodel. Prior to evaluating and testing, respectively, this humananalogue rat model using bisoprolol and the various compounds of thepresent invention, more particularly compounds of formula Ic, theanimals were treated as follows.

In order to generate anti-β1-receptor antibodies the animals wereimmunized against the second β1-receptor loop. In animals neitherreceiving bisoprolol nor any peptide according to the present invention,progressive dilated immune cardiomyopathy was observed after 8 months ofimmunization (FIG. 6). In both the prevention and the therapy study allof the immunized animals developed high titers of stimulatoryanti-β1-EC_(II) antibodies. The specific anti-β1-EC_(II) titer reached amaximum between 6 and 9 months of continuously boosting the animalsevery 4 weeks (FIGS. 4 and 8).

EXAMPLE 3 Prevention of Immune-Cardiomyopathy in the Human Analogue RatModel Using Either Bisoprolol or a Peptide According to the PresentInvention

Prophylactic treatment of the animals by administering 1 mg/kg of thecyclic peptide of formula Ic either subcutaneously (n=4) orintravenously (n=4) every 4 weeks prevented a further increase of thespecific anti-β1-EC_(II) antibody-titer with the titer even continuouslydeclining in the further course of the study (FIG. 4).

To the big surprise of the present inventors, from the fifth month on nomore immune response was observed upon antigen boost every 4 weeks asshown in FIG. 4. This means that some sort of immunological tolerance inthe sense of a hyposensitization with consecutive anergy, i.e.suppression or reduced activation of the anti-β1-EC_(II) producing Bcells occurred. Further analysis of the nature of this “immunologicalanergy” revealed an important decrease in antigen-specificantibody-producing splenic B-cells in the presence of the cyclic peptide(FIG. 20), whereas regulatory CD4⁺ T-cells and/or other mechanisms ofT-cell induced tolerance do not obviously account for this unresponsivestate (FIG. 19 a). FIG. 20 shows that the specifically anti-β1-EC_(II)antibody producing B-cells were significantly reduced in the spleen ofanimals treated with 1 mg/kg of the cyclic peptide of formula Ic.

As shown in FIGS. 5 and 12, the cyclic peptide did neither decreaseheart rate nor blood pressure. In contrast thereto, prophylacticadministration of bisoprolol, as also shown in FIGS. 5 and 12, resultedin a significant reduction in heart rate with the significance levelbeing p<0.01. As may be taken from FIG. 6, there is a significantdifference in the echocardiographic phenotype between theanti-β1-EC_(II)-antibody positive animals which have undergoneprophylaxis and those which had no treatment.

EXAMPLE 4 Treatment of Immune-Cardiomyopathy in the Human Analogue RatModel Using Bisoprolol and a Peptide According to the Present Invention

Upon administration of the peptide of the present invention peptide Icevery 4 weeks, a surprisingly rapid decrease in the specific anti-beta1-EC_(II) antibody titer was observed as shown in FIGS. 8 a and b.Compared with peptide-treated animals, the groups receiving bisoprololhad even higher beta1-antibody titers. As shown in FIGS. 10 and 11, withboth the administration of bisoprolol as well as of the peptide of thepresent invention (after immunization and generation of significant LVdilatation) there was no progression as monitored by echocardiographywithin the 12 months during which the animals underwent this kind oftreatment, compared with untreated anti-beta-1-ECII-antibody positiveanimals. Even more surprisingly, there was even a significant regressionof LV dilatation in all peptide-treated animals with β1-ECII-16AAobviously being less effective than β1-ECII-25AA or β1-ECII-18AA (FIG.11), whereas LV-dilatation could not be reversed completely when usingbisoprolol as the sole therapeutic agent.

These results indicate that with bisoprolol alone the disease may bestabilized, or, by using the present invention peptide, even aregression of the cardiomyopathic phenotype may be achieved.

EXAMPLE 5 Administration of a Combination Consisting of a Cyclic Peptideof the Present Invention and Bisoprolol

Both in the rat model as well as in DCM patients the stimulatory, i.e.allosteric effects of β1-EC_(II)-antibodies can be abolished with acardioselective beta blocker (used in accordance with standardmedication). From this it is concluded that β1-(auto)antibody positivepatients will profit more than β1-(auto)antibody negative patients fromthe present invention. This suggests an even higher efficiency of acombination therapy consisting of bisoprolol and a peptide of thepresent invention for anti-beta-1-EC_(II)-antibody positive patients.Supposed additive mechanisms could be, within others, protection fromantibody-induced receptor-downregulation by the peptide together with asynergistic β1-receptor upregulation by the beta-blockers (likebisoprolol or metoprolol, see FIG. 18). For synergistic effects in theanimal model (see FIGS. 10 c, 11 c, and 13).

EXAMPLE 6 Administration of Shorter Variants of the Cyclic Peptide ofthe Present Invention (i.e. 16 Amino-Acid Vs. 18 Amino-Acid Vs. 25Amino-Acid Cyclic Peptides)

In general, the number of amino acids and thus the length of the primarystructure appears to be crucial for the biological effects of thepeptides of the present invention. A peptide-length equal to or above 26amino acids (primary structure) is thought to be capable of stimulatingdirectly (that is, without the use of carrier proteins) immunocompetentT-cells and thus provoke a paradoxal increase in anti-β1-receptorantibody production through T-cell mediated B-cell stimulation.Therefore, in the frame of the therapy study a limited number of pilotanimals was treated with shorter peptide-variants of the 25 amino-acidpeptide Ic, i.e. variants comprising either 18 or 16 amino-acids of thecyclic peptide. The used constructs were: ECII-18AA,cyclo(Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Gln)(SEQ ID NO:1); and ECII-16AA,cyclo(Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Tyr-Gln)(SEQ ID NO.2). Upon administration of shorter peptide-variants of thepresent invention peptide every 4 weeks, a rapid decrease in thespecific anti-beta 1-EC_(II) antibody titer was equally observed withinthe first 6 months of therapy. Whereas in the following months thecirculating anti-β1-EC_(II) antibody titers were reduced by about a sameextent with either the 25 AA cylic peptide Ic or the 18 AA construct(see FIG. 8 b), both yielding also a similar biological efficiency (i.e.reversal of the cardiomyopathic phenotype; see FIG. 10 d), the 16 AAconstruct tended to be less effective with regard to both, thecontinuous reduction of circulating anti-β1-EC_(II) antibody titers (seegrey diamonds, FIG. 8 b: upon peptide injection the antibody-titersremain stable instead of decreasing) and biological efficiency (i.e.ambiguous results in 2 treated rats with one animal having completereversal of the cardiomyopathic phenotype, the other animal progressiveLV dilatation and dysfunction; see FIGS. 10 d and 11 d). Thesepreliminary results indicate that a certain length of the cyclicreceptor-homologous peptides seems to be necessary to obtain thebeneficial biological effects. However, it should be noted that with the16 AA at least the progression of the disease was stopped or stabilized(comparable with the effects of a monotherapy with beta-blockingagents), as monitored by echocardiography within the 12 months duringwhich the animals underwent this kind of treatment, compared withuntreated anti-beta-1-EC_(II)-antibody positive animals (FIG. 10 d).

EXAMPLE 7 Diagnostic Assay for β-Receptor Antibodies Using Camp System

This detection of β-receptor antibodies (anti-β₁-Abs) in body fluids isbased on the measurement of the impact of antibodies on the β receptormediated signal transduction by optical detection of changes in theconcentration of the intracellular messenger cyclic adenosinemonophosphate (cAMP). Such a cAMP test system is, in principle,described in PCT-application WO 2005/052186.

To detect anti-β₁-Abs by their ability to induce β₁-receptor-mediatedincreases in cAMP, a new highly sensitive cAMP sensor (Epac1-camps)using fluorescence resonance energy transfer is employed. With thistechnology, a total of 56 patient sera and 40 control sera of acollective were analyzed using this method, whereby the patientcollective is the same as already published by Jahns et al. in 1999 andassayed using peptide immuno assay (ELISA) and ¹²⁵iodine labelled cAMPassays for β-receptor antibodies (Jahns et al., Circulation 1999,supra). All those patients which had been β-receptor antibody-positive(n=17) in this earlier study, also showed a significant Epac-1 signal(49±4%). IgG from controls and from about half (17/39) of DCM patientspreviously judged anti-β₁-EC_(II)-negative did not significantly affectcellular cAMP.

Surprisingly, the new technology detected anti-β₁-Abs in more than half(22/39) of DCM patients formerly judged antibody-negative, but the cAMPsignals induced by these antibodies were generally lower (31±5%) than inthe previous group.

Betablockers failed to fully prevent the antibody-induced β₁-receptoractivation, with carvedilol being more effective than other β-blockers.Blocking experiments demonstrated that “high” or “low” activator IgGwere blocked better by peptides corresponding to the second or firstextracellular β1-receptor-loop, respectively.

Taken together, the analysis of Epac-FRET signals from all 56 patientsrevealed an anti-beta 1-Ab prevalence of almost 70% (n=39/56) in DCM,which is much higher than detected by ELISA, immunofluorescence, andcAMP-RIA which proves the suitability of the peptides according to thepresent invention for the diagnosis of DCM. The Abs+ group can beseparated into two subgroups on the basis of the FRET-data, which wereclassified as “low” (FRET amplitude 20-40% of isomax) and “high”activators (FRET amplitude >40% of isomax).

The two newly identified FRET-positive populations were studied in moredetail. Analyzing the concentration-response relation we found that“low” activator IgG, even at higher concentrations, did not achieve cAMPlevels of a similar order of magnitude as induced by “high” activatorIgG (FIG. 23). This finding makes lower titers of anti-beta1-Abs in seraof “low” activators as a possible explanation for the “lower” cAMPresponse rather unlikely, and suggests a differential mechanism ofaction of this type of anti-beta1-Abs at the receptor level.

One reason for qualitative differences between antibodies in terms ofFRET activity might be differences in the receptor-epitopes targeted bydifferent anti-β₁-Abs. Previously functional anti-β₁-Abs against theβ₁-EC_(II) and β₁-EC_(I) epitopes have also been generated successfullyin animals by immunization, suggesting a certain antigenicity of thesetwo epitopes (Mobini R, et al., J. Autoimmun. 1999, 13:179-186). Toanalyze whether different targeted epitopes might account for “high” or“low” FRET-activating capacities of anti-β₁-Abs, we incubated them withsynthetic peptides corresponding to β₁-EC_(II) or to β₁-EC_(I), andanalyzed the blocking effect of each of these peptides. These blockingexperiments revealed that FRET-signals of “high” activator IgG could beattenuated by peptides corresponding to β₁-EC_(II), but not byβ₁-EC_(I)-peptides (7 patients tested; FIG. 24). In contrast, “low”activator IgG were only inhibited by peptides corresponding to β₁-EC_(I)(8 patients tested; FIG. 24). All sera from DCM patients withanti-β₁-Abs yielding cAMP signals near the cut-off value between “high”and “low” activators were studied in such blocking experiments toascertain the accuracy of our classification. The three previouslyAbs-judged patients exhibiting strong Epac-FRET signals (FRET responsebetween 40-60%) were confirmed as “high” activator anti-β₁-EC_(II) Abs,whereas all “low” activator IgG tested (yielding Epac-FRET responsesbetween 20-40%) were inhibited by the β₁-EC_(I) peptide. These data areconsistent with the hypothesis that the differences in cAMP productionof “high” and “low” activators most likely rely on the differentepitopes targeted, and thus on different active receptor conformationsinduced. To address the question whether the differences in type andactivating capacity of anti-β₁-Abs might be relevant for the clinicalcourse of heart failure due to DCM we employed multivariable Coxregression in a retrospective analysis on all-cause mortality in the 56DCM patients analyzed here over a follow-up period of 10 years. FIG. 4depicts the survival curves grouped according to “no”, “low”, and “high”activators, adjusted for age, sex, New York Heart Association functionalclass, hemodynamic status, and medication. The mortality risk in thegroup of “low” activators was not significantly different from patientsnegative for activating anti-β₁-Abs. In contrast, “high” activators hada significantly higher mortality than “low” activators, suggesting thatanti-β₁-EC_(II) Abs are associated with a worse prognosis in DCM. Thiscorroborates the potential pathophysiological and clinical relevance ofactivating anti-beta 1-Abs in heart failure.

In this assay for antibodies directed against β-adrenergic receptors theantibody induced cAMP production is measured in single cells such as,for example, HEK 293 cells or CHW cells which express the β-adrenergicreceptor preferably in a concentration of 0.15 b is 0.3 pmol/mg membraneprotein and are transiently transfected with an Epac-1 sensor.

The Epac-1 sensor as used is a fusion protein of Epac (exchangeprotein-1 directly activated by cAMP) and the binding domain E157-E316coupled to a yellow fluorescent protein (YFP) and the cyan fluorescentprotein (CFP) as described in the paper of Nikolaev et al. (JBC. 2004,279 (36), 37215-8).

These cells are transfected preferably with 10 μg Epac-1 sensor/10 cmdish (diameter) using preferably calcium phosphate precipitation. Themedium is preferably changed 12 to 18 hours after transfection.Preferably, 24 to 48 hours after transfection the antibody-inducedreceptor-mediated signal transduction is determined by measuring thechanges in the cellular cAMP titre using the fluorescence resonanceenergy transfer described in Nikoaev et al. (supra).

The assay is preferably performed using serum samples of patientssuffering from dilated cardiomyopathy and healthy controls. The IgGantibodies are preferably isolated from the serum using caprylic acidmethod, as described in the paper of Jahns et al. (Eur. J. Pharmacol.,1996, 113, 1419-1429). Prior to use the samples are preferably dilutedwith PBS buffer 1:6. Upon addition of the antibodies an increase of theintracellular cAMP level can be detected after about 100 to about 150seconds, as expressed by a decrease in the fluorescence signal generatedby the binding of cAMP to the Epac-1 sensor (corresponding to activationof the receptor). The antibody-induced value is calculated as percentageof the value obtained upon addition of 5 μM of the β-adrenergic agonistisoproterenol.

Apart from that, using this particular assay two further antibodypositive patient groups were identified: three patients had a receptorantibody having a similar effect on intracellular cAMP-increase (44±2%)and another 19 patients had Epac-1-FRET-positive antibodies which had asignificantly lower effect on cAMP-increase (31±5%). Blockingexperiments using epitope-homologous peptides showed that the antibodiesof the first group are directed against the second extracellularβ1-receptor loop, whereas the antibodies of the second group aredirected against the first extracellular β1-receptor loop.

This novel method of detecting anti-beta1-Abs proved to be much fasterand more sensitive than previous methods and is additionally suitable toidentify functionally active β-receptor antibodies against variousreceptor domains. It also revealed an insufficient ability of β-blockersto prevent the anti-beta1-Ab-induced receptor activation. It opens newvenues for the research on anti-beta1-Abs in heart failure and eventualtreatment options.

The features of the present invention disclosed in the specification,the claims and/or the drawings may both separately and in anycombination thereof be material for realizing the invention in variousforms thereof.

1-50. (canceled)
 51. A method for diagnosing a heart disease in asubject comprising: A) introducing to a sample from the subject one ormore peptides selected from the group consisting of: a) a cyclic peptideof formula I: (SEQ ID NO: 3)cyclo(Ala-x-x-x-x-x-x-x-x-x-Cys-x-x-x-Pro-x-Cys- Cys-x_(k)-Gln),

wherein k is any integer from 0 to 6; and wherein x is any amino acid;b) a cyclic peptide of formula II: (SEQ ID NO: 4)Cyclo(Ala-x-x-Trp-x-x-Gly-x-Phe-x-Cys-x_(h)-Gln),

wherein h is any integer from 0 to 2; and wherein x is any amino acid;c) a cyclic peptide of formula III: (SEQ ID NO: 5)Cyclo(Ala-x-x-x-x-x-x-x-x-x-Cys-x_(j)-Cys-x-x-x-Pro-x-Cys-Cys-x_(i)-Gln),

wherein j is any integer from 0 to 4; wherein i is any integer from 0 to6; and wherein x is any amino acid; and d) a peptide of formula IV:(SEQ ID NO: 6) Ala-x_(l)-Cys-x_(m)-Cys-x-x-x-Pro-x-Cys-Cys-x_(n)-Gln,

wherein 1 is any integer from 0 to 9; wherein m is any integer from 0 to4; wherein n is any integer from 0 to 6; and wherein x is any aminoacid; B) performing an assay to detect levels of one or more proteinsthat interact with the one or more peptides in the sample; and C)assessing a risk of or presence of the heart disease in the subject. 52.The method according to claim 51, wherein x is any naturally occurring Lamino acid.
 53. The method according to claim 51, wherein one of the oneor more peptides is a cyclic peptide of formula Ia: (SEQ ID NO: 7)cyclo(Ala-x₂-x-x₁-x-x₁-x₁-x-x₂-x₂-Cys-x-x-x₁-Pro-x- Cys-Cys-x_(k)-Gln),

wherein: x₁ is an acidic amino acid; and x₂ is a basic amino acid. 54.The method according to claim 51, wherein the peptide is a peptide offormula Ic: (SEQ ID NO: 9)cyclo(Ala-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn- Arg-Gln).


55. The method according to claim 51, wherein one of the one or morepeptides is a cyclic peptide of formula IIa: (SEQ ID NO: 10)Cyclo(Ala-x₄-x₂-Trp-x₁-x₃-Gly-x₄-Phe-x₃-Cys-x_(n)-Gln),

wherein n is any integer from 0 to 2; and wherein: x₁ is any acidicamino acid; x₂ is any basic amino acid; x₃ is selected from the groupconsisting of Leu, Ile, Val, Met, Trp, Tyr and Phe; and x₄ is selectedfrom the group consisting of Ser, Thr, Ala and Gly.
 56. The methodaccording to claim 55, wherein one of the one or more peptides is apeptide of formula IIb: (SEQ ID NO: 11)cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys- Glu-Leu-Gln)

or a peptide of formula IIc: (SEQ ID NO: 12)cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe- Cys-Gln).


57. The method according to claim 51, wherein one of the one or morepeptides is a cyclic peptide of formula IIIa: (SEQ ID NO: 13)Cyclo(Ala-x₄-x₂-x₃-x₁-x₃-x₃-x₄-x₃-x₃-Cys-x_(j)-Cys-x-x-x-Pro-x-Cys-Cys-x_(i)-Gln),

wherein i is any integer from 0 to 6; wherein j is any integer from 0 to4; and wherein: x₁ is any acidic amino acid; x₂ is any basic amino acid;x₃ is selected from the group consisting of Leu, Ile, Val, Met, Trp, Tyrand Phe; and x₄ is selected from the group consisting of Ser, Thr, Alaand Gly.
 58. The method according to claim 51, wherein one of the one ormore peptides is a peptide of formula IIIb: (SEQ ID NO: 14)Cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gln).


59. The method according to claim 51, wherein one of the one or morepeptides is a linear peptide of formula IVa: (SEQ ID NO: 15)Ala-x₄-x₂-x₃-x₁-x₃-x₃-x₄-x₃-x₃-Cys-x_(m)-Cys-x-x-x-Pro-x-Cys-Cys-x_(n)-Gln),

wherein n is any integer from 0 to 6; wherein m is any integer from 0 to4; and wherein x₁ is an acidic amino acid; x₂ is a basic amino acid; x₃is selected from the group consisting of Leu, Ile, Val, Met, Trp, Tyrand Phe; and x₄ is selected from the group consisting of Ser, Thr, Alaand Gly.
 60. The method according to claim 59, wherein one of the one ormore peptides is a peptide of formula IVb: (SEQ ID NO: 16)Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gln.


61. The method according to claim 51, wherein cyclization of the cyclicpeptides occurs by a covalent binding linkage selected from the groupconsisting of S—S linkages, peptide bonds, carbon, ester bonds, etherbonds, azo bonds, C—S—C linkages, C—N—C linkages and C═N—C linkages. 62.The method according to claim 61, wherein the S—S linkage is formed bytwo Cys residues of the peptide.
 63. The method according to claim 61,wherein the covalent linkage is a peptide bond formed by the NH2 groupof the N-terminal amino acid and the COOH group of the C-terminal aminoacid.
 64. The method according to claim 51, wherein additional bonds areformed by NH₂ and COOH groups present in side chains of constituentamino acids of the one or more peptides.
 65. The method according toclaim 51, wherein the heart disease is selected from the groupconsisting of infectious and non-infectious heart disease, ischemic andnon-ischemic heart disease, inflammatory heart disease and myocarditis,cardiac dilatation, idiopathic cardio-myopathy, idiopathic dilatedcardiomyopathy, immune-cardiomyopathy, heart failure, and any cardiacarrhythmia including ventricular and supraventricular premature capturebeats.
 66. The method according to claim 65, wherein the heart diseaseis idiopathic dilated cardiomyopathy.
 67. The method according to claim65, wherein the one or more peptides bind to anti-β-adrenergic receptor(anti-β-AR) antibodies and the heart disease is anti-β-AR induceddilated immune-cardiomyopathy.
 68. The method according to claim 51,wherein the method is a FRET-based method.
 69. A method for diagnosing aheart disease in a subject comprising: 1) introducing to a sample fromthe subject one or more peptides selected from the peptides consistingof a cyclic peptide of formula Ib: (SEQ ID  NO: 8)Cyclo(x₄-x₂-x₄-x₁-x₄-x₁-x₁-x₄-x₂-x₂-Cys-x₃-x₅-x₁-Pro-x₂-Cys-Cys-x₁-x₃-x₃-x₄-x₅-x₂-x₅)

wherein: x₁ is any acidic amino acid; x₂ is any basic amino acid; x₃ isselected from the group consisting of Leu, Ile, Val, Met, Trp, Tyr andPhe; x₄ is selected from the group consisting of Ser, Thr, Ala and Gly;and x₅ is selected from the group consisting of Gln and Asn; 2)performing an assay to detect levels of one or more proteins thatinteract with the one or more peptides in the sample; and 3) assessingrisk of or presence of heart disease in the subject.
 70. The methodaccording to claim 69, wherein the heart disease is idiopathic dilatedcardiomyopathy.
 71. The method according to claim 70, wherein the one ormore peptides bind to anti-β-adrenergic receptor (anti-β-AR) antibodiesand the heart disease is anti-β-AR induced dilatedimmune-cardiomyopathy.
 72. The method according to claim 69, wherein themethod is a FRET-based method.